Potential Metabolic Activation of a Representative C4-Alkylated Polycyclic Aromatic Hydrocarbon Retene (1-Methyl-7-isopropyl-phenanthrene) Associated with the Deepwater Horizon Oil Spill in Human Hepatoma (HepG2) Cells

Abstract

Exposure to petrogenic polycyclic aromatic hydrocarbons (PPAHs) in the food chain is the major human health hazard associated with the Deepwater Horizon oil spill. C4-Phenanthrenes are representative PPAHs present in the crude oil and could contaminate the seafood. We describe the metabolism of a C4-phenanthrene regioisomer retene (1-methyl-7-isopropyl-phenanthrene) in human HepG2 cells as a model for metabolism in human hepatocytes. Retene because of its sites of alkylation cannot be metabolized to a diol-epoxide. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. O-Monosulfonated-retene-catechols were discovered as signature metabolites of the ortho-quinone pathway of PAH activation catalyzed by aldo-keto reductases. We also found evidence for the formation of bis-ortho-quinones where the two dicarbonyl groups were present on different rings of retene. The identification of O-monosulfonated-retene-catechol and O-bismethyl-O-monoglucuronosyl-retene-bis-catechol supports metabolic activation of retene by P450 and aldo-keto reductase isozymes followed by metabolic detoxification of the ortho-quinone through interception of redox cycling by catechol-O-methyltransferase, uridine 5'-diphospho-glucuronosyltransferase, and sulfotransferase isozymes. We propose that catechol conjugates could be used as biomarkers of human exposure to retene resulting from oil spills.

Figure 1

HPLC detection of retene metabolites in human HepG2 cells. (A) UV chromatogram at λ_max 258 nm at 0 h. (B) UV chromatogram at λ_max 258 nm at 24 h. (C) FLR chromatogram at λ_ex 259 nm and λ_em 370 nm at 0 h. (D) FLR chromatogram at λ_ex 259 nm and λ_em 370 nm at 24 h. Human HepG2 cells (~5 × 10⁶) were treated with retene (1 μM, 0.2% (v/v) DMSO) in MEM (without phenol red) containing 10 mM glucose. The cell media were collected at 0 and 24 h, respectively, and subsequently acidified with 0.1% formic acid before extraction with ethyl acetate. The extracts were analyzed by HPLC-UV-FLR detection.

Figure 2

Detection of monodehydrated O-monosulfonated-retene-dihydrodiol in human HepG2 cells. (A) MS³ chromatogram at 0 h. (B) MS³ chromatogram at 24 h. (C) MS² spectrum of the peak at 22.80 min. (D) MS³ spectrum of the peak at 22.80 min. The samples were prepared as described in the legend to Figure 1 and were subsequently analyzed on an ion trap LC–MS/MS in the negative ion mode.

Figure 3

Detection of either an O-monosulfonated-retene-catechol or an O-monosulfonated-retene-bis-phenol in human HepG2 cells. (A) MS³ chromatogram at 0 h. (B) MS³ chromatogram at 24 h. (C) MS³ spectrum of the peak at 17.31 min. (D) MS³ spectrum of the peak at 22.79 min. The samples were prepared as described in the legend to Figure 1 and were subsequently analyzed on an ion trap LC–MS/MS in the negative ion mode.

Figure 4

Detection of either O-monoglucuronosyl-retene-catechols or O-monoglucuronosyl-retene-bis-phenols in human HepG2 cells. (A) Extracted ion chromatogram of Orbitrap full scan at 0 h. (B) Extracted ion chromatogram of Orbitrap full scan at 24 h and MS spectrum of the peak at 17.79 min. The samples were prepared as described in the legend to Figure 1 and were subsequently analyzed on an Orbitrap LC–MS/MS in the negative ion mode.

Figure 5

Detection of retene-bis-diones in human HepG2 cells. (A) Extracted ion chromatogram of Orbitrap full scan at 0 h. (B) Extracted ion chromatogram of Orbitrap full scan at 24 h and MS spectrum of the peak at 19.51 min. The samples were prepared as described in the legend to Figure 1 and were subsequently analyzed on an Orbitrap LC–MS/MS in the negative ion mode.

Figure 6

Detection of O-bismethyl-O-monoglucuronosyl-retene-bis-catechols in human HepG2 cells. (A) Extracted ion chromatogram of Orbitrap full scan in the positive mode at 0 h. (B) Extracted ion chromatogram of Orbitrap full scan in the positive mode at 24 h and MS spectrum of the peak at 20.29 min. (C) Extracted ion chromatogram of Orbitrap full scan in the negative mode at 0 h. (D) Extracted ion chromatogram of Orbitrap full scan in the negative mode at 24 h and MS spectrum of the peak at 18.94 min. The samples were prepared as described in the legend to Figure 1 and were subsequently analyzed on an Orbitrap LC-MS/MS.

Scheme 1

Possible Metabolic Activation Pathways of Retene

Scheme 2. Proposed Metabolic Pathway of Retene in Human HepG2 Cells^a

^aNumbers for each metabolite correspond to the metabolites labeled in the UV and fluorescence chromatograms in Figure 1.